Fibronectin is an abundant glycoprotein that is evolutionarily conserved and broadly distributed among vertebrates (Hynes et al., “Fibronectins: Multifunctional Modular Glycoproteins,” J. Cell Biol. 95:369-377 (1982)). Soluble fibronectin is composed of two nearly identical subunits that are joined by disulfide bonds (Petersen et al., “Partial Primary Structure of Bovine Plasma Fibronectin: Three Types of Internal Homology,” Proc. Natl. Acad. Sci. U.S.A. 80:137-141 (1983)). The primary structure of each subunit is organized into three types of repeating homologous units, termed types I, II, and III. Fibronectin type III repeats are found in a number of extracellular matrix (ECM) proteins and consist of two overlapping β sheets (Bork et al., “Proposed Acquisition of an Animal Protein Domain by Bacteria,” Proc. Natl. Acad. Sci. U.S.A. 89(19):8990-8994 (1992) and Leahy et al., “Structure of a Fibronectin Type III Domain From Tenascin Phased by MAD Analysis of the Selenomethionyl Protein,” Science 258:987-991 (1992)). Molecular modeling and atomic force microscopy studies predict that reversible unfolding of the type III repeats contributes to the remarkable elasticity of fibronectin, which may be extended up to six times its initial length without denaturation (Erickson, “Reversible Unfolding of Fibronectin Type III and Immunoglobulin Domains Provides the Structural Basis for Stretch and Elasticity of Titin and Fibronectin,” Proc. Natl. Acad. Sci. 91:10114-10118 (1994) and Oberhauser et al., “The Mechanical Hierarchies of Fibronectin Observed with Single-Molecule AFM,” J. Mol. Biol. 319(2):433-447 (2002)). In the ECM, fibronectin is organized as an extensive network of elongated, branching fibrils. The three-dimensional organization of ECM fibronectin likely arises from the ability of cells to repeatedly exert a mechanical force (Balaban et al., “Force and Focal Adhesion Assembly: A Close Relationship Studied Using Elastic Micropatterned Substrates,” Nat. Cell Biol. 3(5):466-472 (2001)) on discrete regions of the protein (Erickson, “Reversible Unfolding of Fibronectin Type III and Immunoglobulin Domains Provides the Structural Basis for Stretch and Elasticity of Titin and Fibronectin,” Proc. Natl. Acad. Sci. 91:10114-10118 (1994)) to facilitate the formation of fibronectin-fibronectin interactions (Mao et al., “Fibronectin FibrilloGenesis, a Cell-Mediated Matrix Assembly Process,” Matrix Biol. 24(6):389-399 (2005)). As cells contact fibronectin fibrils, tractional forces induce additional conformational changes (Baneyx et al., “Coexisting Conformations of Fibronectin in Cell Culture Imaged Using Fluorescence Resonance Energy Transfer,” Proc. Natl. Acad. Sci. U.S.A. 98(25):14464-14468 (2001)) that are necessary for both lateral growth and branching of the fibrils (Bultmann et al., “Fibronectin Fibrillogenesis Involves the Heparin II Binding Domain of Fibronectin,” J. Biol. Chem. 273:2601-2609 (1998)).
The polymerization of fibronectin into the ECM is a cell-dependent process that is mediated by coordinated events involving the actin cytoskeleton and integrin receptors (Mao et al., “Fibronectin FibrilloGenesis, a Cell-Mediated Matrix Assembly Process,” Matrix Biol. 24(6):389-399 (2005) and Magnusson et al., “Fibronectin: Structure, Assembly, and Cardiovascular Implications,” Arterio. Thromb. Vasc. Biol. 18:1363-1370 (1998)). Most adherent cells, including epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells, polymerize a fibrillar fibronectin matrix (Hynes et al., “Fibronectins: Multifunctional Modular Glycoproteins,” J. Cell Biol. 95:369-377 (1982)). Recent studies have provided evidence that the interaction of cells with either the soluble or ECM form of fibronectin gives rise to distinct cellular phenotypes (Morla et al., “Superfibronectin is a Functionally Distinct Form of Fibronectin,” Nature 367:193-196 (1994) and Hocking et al., “Stimulation of Integrin-Mediated Cell Contractility by Fibronectin Polymerization,” J. Biol. Chem. 275:10673-10682 (2000)). ECM fibronectin stimulates cell spreading (Gui et al., “Identification of the Heparin-Binding Determinants Within Fibronectin Repeat III1: Role in Cell Spreading and Growth,” J. Biol. Chem. 281(46):34816-34825 (2006)), growth (Sottile et al., “Fibronectin Matrix Assembly Enhances Adhesion-Dependent Cell Growth,” J. Cell Sci. 111:2933-2943 (1998) and Sottile et al., “Fibronectin Matrix Assembly Stimulates Cell Growth by RGD-Dependent and -Independent Mechanisms,” J. Cell Sci. 113:4287-4299 (2000)) and migration (Hocking et al., “Fibronectin Polymerization Regulates Small Airway Epithelial Cell Migration,” Am. J. Physiol. Lung Cell Mol. Physiol. 285:L169-L179 (2003)), as well as collagen deposition (Sottile et al., “Fibronectin Polymerization Regulates the Composition and Stability of Extracellular Matrix Fibrils and Cell-Matrix Adhesions,” Mol. Biol. Cell 13:3546-3559 (2002) and Velling et al., “Polymerization of Type I and III Collagens is Dependent on Fibronectin and Enhanced by Integrins Alpha 11beta 1 and Alpha 2beta 1,” J. Biol. Chem. 277(40):37377-37381 (2002)) and organization (Hocking et al., “Stimulation of Integrin-Mediated Cell Contractility by Fibronectin Polymerization,” J. Biol. Chem. 275:10673-10682 (2000)). Others have shown a role for fibronectin matrix assembly in the deposition of fibrinogen (Pereira et al., “The Incorporation of Fibrinogen Into Extracellular Matrix is Dependent on Active Assembly of a Fibronectin Matrix,” J. Cell Sci. 115(Pt 3):609-617 (2002)), fibrillin (Sabatier et al., “Fibrillin Assembly Requires Fibronectin,” Mol. Biol. Cell 20(3):846-858 (2009)), and tenascin C (Chung et al., “Binding of Tenascin-C to Soluble Fibronectin and Matrix Fibrils,” J. Biol. Chem. 270:29012-29017 (1995)) into the ECM. Fibronectin matrix polymerization stimulates the formation of endothelial ‘neovessels’ in collagen lattices (Zhou et al., “Fibronectin Fibrillogenesis Regulates Three-Dimensional Neovessel Formation,” Genes. Dev. 22(9):1231-1243 (2008)). Moreover, blocking fibronectin matrix polymerization inhibits cell growth (Sottile et al., “Fibronectin Matrix Assembly Enhances Adhesion-Dependent Cell Growth,” J. Cell Sci. 111:2933-2943 (1998) and Mercurius et al., “Inhibition of Vascular Smooth Muscle Growth by Inhibition of Fibronectin Matrix Assembly,” Circ. Res. 82:548-556 (1998)) and contractility (Hocking et al., “Stimulation of Integrin-Mediated Cell Contractility by Fibronectin Polymerization,” J. Biol. Chem. 275:10673-10682 (2000)), alters actin organization (Hocking et al., “Inhibition of Fibronectin Matrix Assembly by the Heparin-Binding Domain of Vitronectin,” J. Biol. Chem. 274:27257-27264 (1999)) and cell signaling (Bourdoulous et al., “Fibronectin Matrix Regulates Activation of RHO and CDC42 GTPases and Cell Cycle Progression,” J. Cell Biol. 143:267-276 (1998)), and inhibits cell migration (Hocking et al., “Fibronectin Polymerization Regulates Small Airway Epithelial Cell Migration,” Am. J. Physiol. Lung Cell Mol. Physiol. 285:L169-L179 (2003)). Together, these studies indicate that fibronectin matrix polymerization plays a key role in establishing the biologically-active extracellular environment required for proper tissue function.
Fibronectin matrix assembly is rapidly up-regulated following tissue injury, while reduced fibronectin matrix deposition is associated with abnormal wound repair (Hynes R O, FIBRONECTINS, (Springer-Verlag 1990)). Altered fibronectin matrix deposition is also associated with a large number of chronic diseases including asthma, liver cirrhosis, and atherosclerosis (Hynes R O, FIBRONECTINS, (Springer-Verlag 1990); Roberts C R, “Is Asthma a Fibrotic Disease?” Chest 107:111S-117S (1995); and Stenman et al., “Fibronectin and Atherosclerosis,” Acta. Medica. Scandinavia (Supplement) 642:165-170 (1980)). Given the role of the fibronectin matrix in orchestrating ECM organization and in regulating cell and tissue responses critical for tissue repair, defective or diminished fibronectin matrix deposition by cells is likely to have profound effects on the ability of tissues to heal.
The present invention is directed to overcoming these and other deficiencies in the art.